Rabbit monoclonal antibodies to hepatitis B surface antigens and methods of using the same

ABSTRACT

Reagents, methods and immunodiagnostic test kits for the accurate detection of hepatitis B virus (HBV) infection are disclosed. The methods and kits employ novel rabbit monoclonal antibodies directed against HBV surface antigens (HBsAg) with mutations in the “a” determinant region of HBsAg.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of provisionalapplication Ser. No. 60/577,561, filed on Jun. 7, 2004, and provisionalapplication Ser. No. 60/583,734, filed on Jun. 28, 2004, whichapplications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention pertains generally to hepatitis B virus (HBV). Inparticular, the invention relates to rabbit monoclonal antibodiesdirected against HBV surface antigens and methods of use thereof fordiagnosis of HBV infection.

BACKGROUND

Hepatitis B virus (HBV) is a member of a group of small DNA-containingviruses that cause persistent noncytopathic infections of the liver. HBVinfection in humans can cause severe jaundice, liver degeneration anddeath. HBV enters predominantly by the parenteral route, has acharacteristic incubation period of 60 to 160 days, and may persist inthe blood for years in chronic carriers. HBV is of great medicalimportance because it is one of the most common causes of chronic liverdisease, such as hepatocellular carcinoma, in humans. Infectedhepatocytes continually secrete viral particles that accumulate to highlevels in the blood. Moreover, it is estimated that about 6 to 7% of thehuman population is infected, with the level of infection being as highas 20% of the population in certain regions of Southeast Asia andsub-Sahara Africa.

Several tests have been employed to detect the presence of HBVconstituents in serum and other body fluids. These tests are primarilyimmunological in principle and depend on the presence of antibodiesproduced in humans or animals to detect specific viral proteins such asthe hepatitis B surface antigen (HBsAg), hepatitis B core (nucleocapsid)antigen (HBcAg) or hepatitis B “E” antigen (HBeAg). However, there areincreasing concerns about the contribution of variant HBsAgs relative tothe production of false negatives in serological HBsAg diagnosis orblood screening assays.

In particular, HBV, due to its mode of replication by reversetranscription of its pre-genomic RNA, has a high rate of mutationrelative to other DNA viruses. Amino acid substitutions have beendescribed in all HBV DNA-encoded viral proteins such as polymerase,HBcAg and HBsAg. The group-specific “a” determinant region of HBV (aminoacids 124-147, numbered relative to the S portion of HBsAg) hasattracted the most attention, because mutations in this region have beenfound in 10-20% of vaccine escapees and have resulted in themisdiagnoses of variant HBVs, even using the most current serologicalassays on the market. Thus, there is a need for the development ofreliable diagnostic tests to detect HBV in viremic samples, in order toprevent transmission of the virus through blood and plasma derivativesor by close personal contact.

Rabbit-rabbit and rabbit-mouse hybridomas have been used in an attemptto generate monoclonal antibodies with increased immunoreactivity. See,e.g., U.S. Pat. Nos. 4,977,081; 4,859,595; 5,472,868; 5,675,063;Spieker-Polet et al., Proc. Natl. Acad. Sci. USA (1995) 92:9348-9352.Rabbit monoclonal antibodies are desirable for several reasons. First,rabbits may recognize antigens and epitopes that are not immunogenic inmice or rats, the two species from which monoclonal antibodies areusually generated. Additionally, rabbit antibodies are generally of highaffinity. U.S. Pat. No. 4,859,595 describes the production of rabbitmonoclonal antibodies to HBsAg using rabbit-rabbit fusions.

However, there remains a need for improved immunoassays using monoclonalantibodies with broader immunoreactivity against the various HBsAgmutants. The wide-spread availability of reagents for use in an accurateand efficient assay for HBV infection would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides highly immunoreactive monoclonalantibodies for the simple, accurate and efficient diagnosis of HBVinfection. The antibodies are produced from rabbit hybridomas and areimmunoreactive against various mutant HBV strains. Thus, assay methodsusing the rabbit monoclonal antibodies are more accurate and the numberof false negatives seen with other serological tests is reduced. Assaysusing the antibodies therefore allow the detection of HBV infectioncaused by a variety of HBV mutants and, if infection is detected, theindividual can be given appropriate treatment in adequate time to helpprevent liver damage and death.

Accordingly, in one embodiment, the invention is directed to an anti-HBVrabbit monoclonal antibody that recognizes an HBsAg mutant with amutation in the “a” determinant region, or an immunoreactive fragmentthereof, such as a Fab, F(ab′)₂, Fv or an sFv fragment. In certainembodiments, the antibody recognizes more than one HBsAg mutant with amutation in the “a” determinant region. In additional embodiments, theantibody also recognizes a wild-type HBsAg. In yet further embodiments,the HBsAg mutant is a mutant sAg, such as a mutant that comprises thesequence of the “a” determinant region of F134A, F134S, G145R, S143L,P142S or Q129R/M133T. In further embodiments, the antibody recognizes anHBsAg and/or one or at least two mutant HBSAg selected from the groupconsisting of F134A, F134S, G145R, S143L, P142S and Q129R/M133T. Any ofthe antibodies above can be produced using a rabbit-rabbit hybridoma ora rabbit-mouse hybridoma.

In additional embodiments, the invention is directed to hybridomas 99S6(ATCC Accession number PTA-6015) and 99S9 (ATCC Accession numberPTA-6014), and antibodies produced by these hybridomas. In furtherembodiments, the invention is directed to a rabbit monoclonal antibodythat recognizes the same epitope as an antibody produced by hybridoma99S6 and/or 99S9.

In further embodiments, the invention is directed to a method ofdetecting HBV surface antigens in a biological sample. The methodcomprises:

contacting the biological sample with at least one rabbit monoclonalantibody according to any of the embodiments above, under conditionswhich allow HBV antigens, when present in the biological sample, to bindto the antibody to form an antibody/antigen complex; and

detecting the presence or absence of the antibody/antigen complex,

thereby detecting the presence or absence of HBV surface antigens in thesample.

In preferred embodiments of the above method, the at least one rabbitmonoclonal antibody is the antibody produced by the hybridoma 99S6 orthe hybridoma 99S9. In certain embodiments, the at least one rabbitmonoclonal antibody is detectably labeled. In certain embodiments, themethod further comprises reacting the biological sample with one or moreadditional antibodies directed against a wild-type HBsAg or an HBsAgmutant with a mutation in the “a” determinant region. The one or moreadditional antibodies may comprise an additional monoclonal antibody,such as a mouse monoclonal antibody.

In additional embodiments, the invention is directed to animmunodiagnostic test kit for detecting HBV infection. The test kitcomprises:

at least one rabbit monoclonal antibody, or immunoreactive fragmentthereof according to any of the embodiments above; and

instructions for conducting the immunodiagnostic test.

In preferred embodiments of the above method, the at least one rabbitmonoclonal antibody is the antibody produced by the hybridoma 99S6 orthe hybridoma 99S9. In certain embodiments, the test kit furthercomprises one or more additional antibodies directed against a wild-typeHBsAg or an HBsAg mutant with a mutation in the “a” determinant region.The one or more additional antibodies may comprise an additionalmonoclonal antibody, such as a mouse monoclonal antibody.

In further embodiments, the invention is directed to a solid supportcomprising at least one rabbit monoclonal antibody or immunoreactivefragment thereof according to any of the above embodiments. In preferredembodiments of the solid support, the at least one rabbit monoclonalantibody is the antibody produced by the hybridoma 99S6 or the hybridoma99S9. In certain embodiments, the support further comprises one or moreadditional antibodies directed against a wild-type HBsAg or an HBsAgmutant with a mutation in the “a” determinant region. The one or moreadditional antibodies may comprise an additional monoclonal antibody,such as a mouse monoclonal antibody. In additional embodiments, thesolid support further comprises at least two internal controls, whereinone of the controls defines the lower detection limit for a positiveresult in an immunoassay using the solid support and the other controldefines a highly positive result in an immunoassay using the solidsupport. In some embodiments, the solid support is a nitrocellulosestrip.

In yet additional embodiments, the invention is directed to animmunodiagnostic test kit for detecting HBV. The test kit comprises:

(a) a solid support according to any of the above embodiments; and

(b) instructions for conducting the immunodiagnostic test.

In a further embodiment, the invention is directed to a method ofdetecting the presence of HBV surface antigens in a biological sample.The method comprises:

(a) providing a biological sample;

(b) providing a solid support as described above;

(c) contacting the biological sample with the solid support, underconditions which allow HBV surface antigens, if present in thebiological sample, to bind with at least one of the rabbit monoclonalantibodies to form an antibody/antigen complex; and

(d) detecting the presence of the antibody/antigen complex,

thereby detecting the presence of HBV surface antigens in the biologicalsample.

In certain embodiments, the method further comprises:

(e) removing unbound HBV antigens;

(f) providing one or more moieties capable of associating with theantibody/antigen complex; and

(g) detecting the presence of the one or more moieties,

thereby detecting the presence of HBV surface antigens in the biologicalsample.

In additional embodiments of the method, the one or more moietiescomprises a detectably labeled HBV antibody, such as a detectablylabeled rabbit monoclonal antibody that recognizes an HBsAg mutant witha mutation in the “a” determinant region, or an immunoreactive fragmentthereof. The detectable label can be an enzyme. Additionally, thebiological sample can be from a human blood sample.

In further embodiments, the invention is directed to a method ofdetecting the presence of anti-HBsAg antibodies in a biological sample.The method comprises:

(a) providing a solid support as described above;

(b) contacting the solid support with one or more HBsAgs, underconditions which allow the one or more HBsAgs to bind with at least oneof the rabbit monoclonal antibodies to form an antibody/antigen complex;

(d) contacting the solid support having the antibody/antigen complexwith a biological sample, under conditions which allow anti-HBsAgantibodies, if present in the biological sample, to bind with theantibody/antigen complex to form an antibody/antigen/antibody complex;and

(e) detecting the presence of the antibody/antigen/antibody complex,

thereby detecting the presence of anti-HBsAg antibodies in thebiological sample.

In further embodiments, the method further comprises:

(f) removing unbound antibodies;

(g) providing one or more moieties capable of associating with theantibody/antigen/antibody complex, such as one or more moietiescomprising a detectably labeled immunoglobulin molecule; and

(h) detecting the presence of the one or more moieties,

thereby detecting the presence of anti-HBsAg antibodies in thebiological sample.

In additional embodiments, the invention is directed to a method ofpreparing a blood supply comprising whole blood, platelets, plasma orserum, substantially free of HBV. The method comprises:

(a) screening aliquots of whole blood, platelets, plasma or serum fromcollected blood samples by a method above;

(b) eliminating any samples in which an HBV antigen is detected; and

(c) combining samples in which no HBV antigen is detected to provide ablood supply substantially free of HBV.

In further embodiments, the invention is directed to a method ofpreparing a blood supply comprising whole blood, platelets, plasma orserum, substantially free of HBV. The method comprises:

(a) screening aliquots of whole blood, platelets, plasma or serum fromcollected blood samples by a method above;

(b) eliminating any samples in which an anti-HBsAg antibody is detected;and

(c) combining samples in which no anti-HBsAg antibody is detected toprovide a blood supply substantially free of HBV.

In yet additional embodiments, the invention is directed to a method ofscreening a donated tissue or organ prior to transplantation to providea tissue or organ substantially free of HBV. The method comprises:

(a) screening a sample from the tissue or organ by a method above;

(b) eliminating a tissue or organ in which an HBV antigen is detected toprovide a tissue or organ substantially free of HBV.

In further embodiments, the invention is directed to a method ofscreening a donated tissue or organ prior to transplantation to providea tissue or organ substantially free of HBV. The method comprises:

(a) screening a sample from the tissue or organ by a method above;

(b) eliminating a tissue or organ in which an anti-HBsAg antibody isdetected to provide a tissue or organ substantially free of HBV.

In still further embodiments, the invention is directed to a method ofpreparing an anti-HBV rabbit monoclonal antibody. The method comprises:

(a) immunizing a rabbit with an HBsAg mutant with a mutation in the “a”determinant region;

(b) fusing cells that produce antibodies against the HBsAg mutant fromthe rabbit with a cell from an immortalized cell line to produce ahybridoma;

(c) selecting for the hybridoma;

(d) culturing the selected hybridoma; and

(e) collecting the antibody secreted by the cultured hybridoma.

In certain embodiments, the immunizing step comprises immunizing arabbit with more than one HBsAg mutant. In certain embodiments, theantibody-producing cells are rabbit splenocytes. The splenocytes can befused with a cell from an immortalized rabbit cell line, such as arabbit plasmacytoma, to produce a rabbit-rabbit hybridoma, or with acell from an immortalized mouse cell line, to produce a rabbit-mousehybridoma.

In certain embodiments, of the method above, the HBsAg mutant is amutant sAg, such as a mutant that comprises the sequence of the “a”determinant region of F134A, F134S, G145R, S143L, P142S or Q129R/M133T.In other embodiments, the HBsAg mutant comprises F134A, F134S, G145R,S143L, P142S or Q129R/M133T. In yet further embodiments, the rabbit isimmunized with at least two HBsAg mutants with different mutations inthe “a” determinant region, such as with at least two HBsAg mutantsselected from the group consisting of F134A, F134S, G145R, S143L, P142Sor Q129R/M133T. In additional embodiments, the rabbit is immunized withHBsAg mutants F134A, F134S, G145R, S143L, P142S and Q129R/M133T. Infurther embodiments, the rabbit is additionally immunized with a wildtype HBsAg.

In additional embodiments, the invention is directed to an anti-HBVrabbit monoclonal antibody produced by the methods above.

In yet further embodiments, the invention is directed to a method ofpreparing a rabbit-rabbit hybridoma. The method comprises:

(a) immunizing a rabbit with an HBsAg mutant with a mutation in the “a”determinant region;

(b) fusing splenocytes that produce antibodies against the HBsAg mutantfrom the rabbit with cells from a rabbit plasmacytoma;

(c) selecting for cells that secrete the antibodies.

In additional embodiments, the invention is directed to a polynucleotideencoding a rabbit monoclonal antibody or an immunoreactive fragmentthereof such as a Fab, F(ab′)₂, Fv or an sFv fragment, as describedabove.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the HBV surface antigen depicting the highlyconformational structure of the protein (lower panel, solid line) andthe amino acid sequence (SEQ ID NO:1) around the “a” determinant (fromaa 121-147, upper panel, in circles). The arrows indicate the positionand substitution of various known HBsAg variants.

FIGS. 2A and 2B (SEQ ID NOS:2 and 3) show the amino acid sequence forthe sAg wild-type adw and ayw antigens, respectively.

FIGS. 3A-3D show the immunoreactivities of rabbit monoclonal antibodiesfrom 99S6 (FIG. 3B) and 99S9 (FIG. 3D), in comparison with the mouseantibodies mMAb1 (FIG. 3C) and mMAb2 (FIG. 3A) against the HBV mutantpanel described above.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, chemistry, biochemistry,recombinant DNA techniques and immunology, within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Fundamental Virology, 3rd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M.Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E.Creighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,Inc., current addition); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

The following amino acid abbreviations are used throughout the text:Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a rabbit monoclonal antibody” includes a mixture of two ormore such polypeptides, and the like.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

The term “antigen” refers to a polypeptide, whether native, recombinantor synthetic, which includes one or more epitopes that recognize anantibody. The antigen in question need not include the full-length aminoacid sequence of the reference molecule but can include only so much ofthe molecule as necessary in order to generate an immunological reaction(i.e., when the antigen is used for generating antibodies) or to reactwith the HBV antibody of interest (i.e., where the antigen is beingdetected in an assay). Thus, only one or few epitopes of the referencemolecule need be present. Furthermore, the antigen may comprise a fusionprotein between the full-length reference molecule or a fragment of thereference molecule, and another protein such as another HBV antigenand/or a protein that does not disrupt the reactivity of the HBVantigen. It is readily apparent that the antigen may therefore comprisethe full-length sequence, fragments, truncated and partial sequences, aswell as analogs, muteins and precursor forms of the reference molecule.The term also intends deletions, additions and substitutions to thereference sequence, so long as the antigen retains the ability tostimulate antibody production and/or to react with HBV antibodies.

In this regard, natural variation will occur from isolate to isolatewithin a particular HBV strain. Thus, the term is intended to encompasssuch variation and, in particular, an antigen that varies in its aminoacid composition by not more than about 20 number percent, morepreferably by not more than about 10 to 15 number percent, and mostpreferably, by not more than about 5 number percent, from the referenceantigen. Proteins having substantially the same amino acid sequence asthe reference molecule, but possessing minor amino acid substitutionsthat do not substantially affect the antibody binding capabilities ofthe antigen, are therefore within the definition of the referencepolypeptide.

An antigen “derived from” an HBV strain or isolate intends an antigenwhich comprises a sequence of one or more regions or portions of regionsof an antigen encoded by the reference HBV genome. Typically, theantigen is composed of regions or portions of regions that includeepitopes, and will generally have an amino acid sequence substantiallyhomologous to the reference polypeptide, as defined below. Thus, theterm “derived from” is used to identify the original source of amolecule but is not meant to limit the method by which the molecule ismade which can be, for example, by chemical synthesis or recombinantmeans.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, that retain desired activity, such asimmunoreactivity in assays described herein. In general, the term“analog” refers to compounds having a native polypeptide sequence andstructure with one or more amino acid additions, substitutions(generally conservative in nature) and/or deletions, relative to thenative molecule, so long as the modifications do not destroy immunogenicactivity and which are “substantially homologous” to the referencemolecule as defined below. A number of conserved and variable regionsare known between the various isolates and, in general, the amino acidsequences of epitopes derived from these regions will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than50%, generally more than 60%-70%, when the two sequences are aligned.The term “mutein” refers to peptides having one or more peptide mimics(e.g., “peptoids”). Preferably, the analog or mutein has at least thesame immunoreactivity as the native molecule. Methods for makingpolypeptide analogs and muteins are known in the art and are describedfurther below.

The terms “analog” and “mutein” also encompass purposeful mutations thatare made to the reference molecule. Particularly preferred analogsinclude substitutions that are conservative in nature, i.e., thosesubstitutions that take place within a family of amino acids that arerelated in their side chains. Specifically, amino acids are generallydivided into four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serinethreonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. For example, it isreasonably predictable that an isolated replacement of leucine withisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar conservative replacement of an amino acid with astructurally related amino acid, will not have a major effect on thebiological activity. For example, the antigen of interest may include upto about 5-10 conservative or non-conservative amino acid substitutions,or even up to about 15-25, 50 or 75 conservative or non-conservativeamino acid substitutions, or any integer between 5-75, so long as thedesired function of the molecule remains intact. One of skill in the artcan readily determine regions of the molecule of interest that cantolerate change by reference to Hopp/Woods and Kyte-Doolittle plots,well known in the art.

By “antigen fragment” is intended an antigen consisting of only a partof the intact full-length antigen polypeptide sequence and structure.The fragment can include a C-terminal deletion, an N-terminal deletion,and/or an internal deletion of the native polypeptide. By “immunogenicfragment” is meant a fragment of a polypeptide that includes one or moreepitopes and thus elicits one or more of the immunological responsesdescribed herein. An “immunogenic fragment” of a particular HBV proteinwill generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains the ability to elicit an immunologicalresponse as defined herein.

By “HBsAg” is meant an HBV surface antigen derived from any of thevarious HBV strains and isolates. The term intends surface antigenswhich include a substantially complete S domain of an HBsAg polypeptide(termed “sAg” herein), as well as immunogenic fragments thereof. An Sdomain of HBsAg is “substantially complete” if it contains the nativesequence of the polypeptide with or without minor deletions of one or afew amino acids from either the N-terminal or C-terminal regions orwithin the polypeptide. For example the HBsAg S domain can be truncatedby a few amino acids, i.e., up to about 3, 5, 7, or 10 amino acids,without greatly affecting its antigenicity. An HBsAg antigen for useherein will generally include a region corresponding to the “a”determinant, found at amino acid positions 124-147, numbered relative tothe sAg. This region is described further below. The term also intendsan antigen that includes the preS2 (formerly called preS) domain inaddition to the S domain, or both the preS2 and preS1 domains of HBsAg,in addition to the S domain. Valenzuela, et al. (1982) Nature298:347-350, describes the gene for a representative HBsAg. See, also,Valenzuela, et al. (1979) Nature 280:815-819.

A “mutant” HBsAg molecule, as used herein, refers to analogs ofwild-type HBsAgs, as defined above. For the purpose of this invention,by “wild-type HBsAgs” is meant HBsAgs from the ayw and adw subtypes.These analogs may arise by natural mutational events, e.g., in the caseof escape mutants, or may be purposefully created. Representative mutantHBsAg sequences are shown in FIG. 1 herein. Additional naturallyoccurring mutants are known in the art and the nucleotide sequences andcorresponding amino acid sequences for surface antigens from thesemutants have been deposited with GenBank. See, e.g., NCBI accessionnumbers AY341335 (naturally occurring surface mutant with multiplemutations in the “a” determinant of sAg), X59795 (naturally occurringmutant from the ayw subtype); AF01360 and AF013629 (naturally occurringmutants from the adw subtype) and Zuckerman et al. 1999 (J. Med Virol.58:193).

By “immunogenic” sequence of an HBsAg is meant an HBsAg molecule thatincludes an amino acid sequence with at least one epitope such that themolecule is capable of stimulating the production of antibodies in anappropriate host. By “epitope” is meant a site on an antigen to whichspecific B cells and/or T cells respond, rendering the HBV epitope inquestion capable of stimulating antibody production. The term is alsoused interchangeably with “antigenic determinant” or “antigenicdeterminant site.” An epitope can comprise 3 or more amino acids in aspatial conformation unique to the epitope. Generally, an epitopeconsists of at least 5 such amino acids and, more usually, consists ofat least 8-10 such amino acids or more.

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

An “immunogenic composition” is a composition that comprises at leastone immunogenic polypeptide (e.g., an HBsAg antigen or antibody).

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different isolates or strains of HBV which antigenic determinantsare not necessarily identical due to sequence variation, but which occurin equivalent positions in the HBV sequence in question. In general theamino acid sequences of equivalent antigenic determinants will have ahigh degree of sequence homology, e.g., amino acid sequence homology ofmore than 30%, usually more than 40%, such as more than 60%, and evenmore than 80-90% homology, when the two sequences are aligned.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50%, preferably at least about 75%, more preferably atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two molecules(the reference sequence and a sequence with unknown % identity to thereference sequence) by aligning the sequences, counting the exact numberof matches between the two aligned sequences, dividing by the length ofthe reference sequence, and multiplying the result by 100. Readilyavailable computer programs can be used to aid in the analysis, such asALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.Dayhoff ed., 5 Suppl. 3:353-358, National biomedical ResearchFoundation, Washington, D.C., which adapts the local homology algorithmof Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 forpeptide analysis. Programs for determining nucleotide sequence identityare available in the Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.) for example, theBESTFIT, FASTA and GAP programs, which also rely on the Smith andWaterman algorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

An “antibody” intends a molecule that “recognizes,” i.e., specificallybinds to an epitope of interest present in an antigen. By “specificallybinds” is meant that the antibody interacts with the epitope in a “lockand key” type of interaction to form a complex between the antigen andantibody, as opposed to non-specific binding that might occur betweenthe antibody and, for instance, components in a mixture that includesthe test substance with which the antibody is reacted. Thus, an anti-HBVantibody is a molecule that specifically binds to an epitope of an HBVprotein. The term “antibody” as used herein includes antibodies obtainedfrom both polyclonal and monoclonal preparations, as well as, thefollowing: hybrid (chimeric) antibody molecules (see, for example,Winter et al., Nature (1991) 349:293-299; and U.S. Pat. No. 4,816,567);F(ab′)2 and F(ab) fragments; Fv molecules (non-covalent heterodimers,see, for example, Inbar et al., Proc Natl Acad Sci USA (1972)69:2659-2662; and Ehrlich et al., Biochem (1980) 19:4091-4096);single-chain Fv molecules (sFv) (see, for example, Huston et al., ProcNatl Acad Sci USA (1988) 85:5879-5883); dimeric and trimeric antibodyfragment constructs; minibodies (see, e.g., Pack et al., Biochem (1992)31:1579-1584; Cumber et al., J Immunology (1992) 149B:120-126);humanized antibody molecules (see, for example, Riechmann et al., Nature(1988) 332:323-327; Verhoeyan et al., Science (1988) 239:1534-1536; andU.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and,any functional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)₂, Fv, and other fragments, as well as chimeric and humanizedhomogeneous antibody populations, that exhibit immunological bindingproperties of the parent monoclonal antibody molecule.

As used herein, the term “rabbit monoclonal antibody” refers to amonoclonal antibody, as defined above, produced by immunizing a rabbitwith an antigen of interest (e.g., a mutant HBsAg). A “rabbit monoclonalantibody” can be produced using rabbit-rabbit hybridomas (i.e., fusionsbetween an antibody-producing cell from the immunized rabbit with animmortalized cell from a rabbit), rabbit-mouse hybridomas (i.e., fusionsbetween an antibody-producing cell from the immunized rabbit with animmortalized cell from a mouse), and the like, described more fullybelow.

A “mouse monoclonal antibody” refers to a monoclonal antibody, asdefined above, produced by immunizing a mouse, with an antigen ofinterest (e.g., a mutant HBsAg). A “mouse monoclonal antibody” isproduced using conventional methods well known in the art, frommouse-mouse hybridomas, described more fully below.

As used herein, a “solid support” refers to a solid surface to which amacromolecule, e.g., an antibody, protein, polypeptide, peptide,polynucleotide can be attached, such as a magnetic bead, latex bead,microtiter plate well, glass plate, nylon, agarose, polyacrylamide,silica particle, nitrocellulose membrane, and the like.

“Immunologically reactive” means that the antibody in question willreact specifically with HBV antigens present in a biological sample froman HBV-infected individual.

An “immunoreactive fragment” of an antibody, is a molecule consisting ofonly a portion of the intact antibody sequence and structure, and thatis immunologically reactive as defined above. Non-limiting examples ofsuch immunoreactive fragments include F(ab′)₂, Fv, and sFv molecules,that are capable of exhibiting immunological binding properties of theparent antibody molecule from which they are derived.

“Immune complex” intends the combination formed when an antibody bindsto an epitope on an antigen.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject such as, but not limited to, blood,plasma, platelets, serum, fecal matter, urine, bone marrow, bile, spinalfluid, lymph fluid, cerebrospinal fluid, samples of the skin, secretionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components. The samples detailed above neednot necessarily be in the form obtained directly from the source. Forexample, the sample can be treated prior to use, such as, for example,by heating, centrifuging, etc. prior to analysis.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, semiconductor nanocrystals,chemiluminescers, chromophores, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands(e.g., biotin, streptavidin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof which is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels which may be used under the invention include, but are notlimited to, horse radish peroxidase (HRP), fluorescein, FITC, rhodamine,dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red,luminol, NADPH and α-β-galactosidase.

II. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention is based on the discovery that novel rabbitmonoclonal antibodies, directed against mutant HBsAgs, are far moreimmunoreactive in assays for detecting HBV infection than conventionalmouse monoclonal antibodies. The rabbit monoclonal antibodies of thepresent invention are reactive with a broader range of HBsAg mutantsthan conventional mouse monoclonal antibodies. Moreover, the rabbitmonoclonal antibodies of the present invention are typically alsoreactive with wild-type HBsAgs. Indeed, a single rabbit monoclonalantibody according to the present invention is as effective as the useof multiple mouse monoclonal antibodies for detecting the presence ofHBV antigens, which can be indicative of HBV infection. Thus, the rabbitmonoclonal antibodies of the present invention decrease the number offalse negatives obtained with assays using, e.g., mouse monoclonalantibodies and are therefore useful in diagnostic methods for accuratelydetecting HBV infection. The assays of the present invention can alsoutilize additional antibodies, such as additional mouse monoclonalantibodies, to provide the ability to diagnose HBV infection from a widevariety of isolates and escape mutants.

The methods are useful for detecting HBV infection in humans, as well asfor detecting HBV infection in blood samples, including withoutlimitation, in whole blood, serum, platelets, and plasma, as well as intissues and organs for transplantation, in particular by detecting thepresence of HBV antigens or HBV antibodies. Thus, the methods can beused to diagnose HBV infection in a subject, such as a human subject, aswell as to detect HBV contamination in donated blood samples. Aliquotsfrom individual donated samples or pooled samples can be screened forthe presence of HBV and those samples or pooled samples contaminatedwith HBV can be eliminated before they are combined. In this way, ablood supply substantially free of HBV contamination can be provided.Similarly, samples from tissues and organs to be used in transplantationcan also be screened in order to eliminate contaminated specimens.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding HBV antigens, antibodies anddiagnostic methods for use with the subject invention.

HBV Surface Antigens

The hepatitis B surface antigens are made up of three size classes ofproteins that share carboxy-terminal sequences. These proteins includelarge (L, the preS2 domain), medium (M, the preS1 domain), and small (S,the sAg domain). All three proteins are found in infectious virions(often referred to as Dane particles) recovered as 42 nm spheres fromthe serum of infected patients. Serum samples also contain emptyspherical particles averaging 22 nm, which contain primarily the S classof proteins (sAg). Mammalian cell lines transfected exclusively with DNAencoding the sAg protein release 20 nm empty spheres similar to thosefrom infected cells. Moreover, yeast cells transformed with the samegene form analogous spheres, which are found to be equally immunogenicas the 22 nm spheres from infected cells. See, e.g., “HBV Vaccines—fromthe laboratory to license: a case study” in Mackett, M. and Williamson,J. D., Human Vaccines and Vaccination, pp. 159-176, for a discussion ofHBV structure; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513,5,965,140, incorporated herein by reference in their entireties, Beameset al., J. Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990)64:3319-3330, Zhou et al., J. Virol. (1991) 65:5457-5464, fordescriptions of the recombinant production of various HBV particles.

Thus, as explained above, HBsAgs for use in producing the rabbitmonoclonal antibodies of the present invention can include immunogenicregions of sAg, preS1 and/or preS2, as well as immunogenic regions fromany combination of the above, such as sAg/preS1, sAg/preS2, andsAg/preS1/preS2. Optionally, an HBsAg polypeptide can comprise more thanone sAg, preS1, or preS2 polypeptide. Additionally, the sAg, preS1, andpreS2 polypeptides may be derived from the same or different isolates ofHBV. These polypeptides may also be provided as a fusion protein or asseparate polypeptides. The sequences of HBsAgs from hundreds ofdifferent HBV isolates are known and can be readily obtained from theNCBI database.

A preferred HBsAg for use in the invention comprises at least thesequence of amino acids of the “a” determinant region of HBV (aminoacids 124-147, numbered relative to the sAg). Representative wild-typesequences for this region are CTTPAQGNSMFPSCCCTKPSDGNC (SEQ ID NO:4, adwwild-type); and CMTTAQGTSMYPSCCCTKPSDGNC (SEQ ID NO:5, ayw wild-type).Mutations in this region of sAg have been found in a large number of HBVvaccine escapees. For descriptions of a number of HBsAg variants, see,Ashton-Richardt P G, Murray K. (1989) Mutants of the hepatitis B virussurface antigen and define some antigenically essential residues in theimmunodominant “a” region. J. Medical Virology 29: 196-203; Norder H.Courouce A_M, Magnius L (1992) Molecular basis of hepatitis B virusserotype variation within the four major subtype. J. General Virology73: 3141-3145; Carman W F, Zanetti A R, et. al. (1990) Vaccine-inducedescape mutant of hepatitis B virus. Lancet 336: 325-329; Fujii H.Moriyama K. et al. Gly 145 to Arg substitution in HBs antigen of immuneescape mutant of hepatitis B virus. Biochem. Biophys Res Comm 184:1152-1157; Carman, W. Vaccine-associated mutants of hepatitis B virus.Viral Hepatitis and Liver Disease (1945) pp: 243-247, Eds: K. Nishioka,H. Suzuki, S. Mishiro T. Oda)

Thus, HBsAgs including mutations in this region are particularly usefulherein. Representative mutants for this region include F134A, F134S,G145R, S143L, P142S and Q129R/M133T. In each of the mutant designations,the number indicates the position of the substituted amino acid, theletter before the number indicates the amino acid at that position inthe WT sequence and the letter following the number indicates the aminoacid at that position in the mutant. These mutants are merelyrepresentative and it is to be understood that a large number ofadditional naturally occurring mutants exist, which mutants will finduse with the present invention. Additionally, synthetic mutants withmutations in the “a” determinant region will also find use herein.Variants having mutations in regions other than the “a” determinantregion, as defined above, may also find use in the present invention.For example, the variant having a substitution of Q for P at amino acidposition 120 (P120Q), finds use as antigen for generating monoclonalantibodies.

Antigens for use with the present invention can be obtained usingstandard techniques. The HBV antigens are conveniently generated usingrecombinant methods, well known in the art. See, e.g., U.S. Pat. Nos.4,722,840, 5098,704, 5324,513, 5,965,140 and 6,306,625, for descriptionsof the recombinant production of HBV antigens, all of which areincorporated herein by reference in their entireties. For example, theHBsAg S protein coding sequence can be isolated by phenol extraction ofDNA from Dane particles present in infected human serum, using methodsknown in the art, such as described in U.S. Pat. No. 4,710,463, thedisclosure of which is incorporated herein by reference in its entirety.The isolated DNA can then be digested with a restriction endonuclease.The choice of endonuclease will depend, in part, on the particular Daneparticles. For example, the HBsAg coding sequence of HBV DNA of certainDane particles of the adw serotype can be isolated as a single BamHIfragment; the HBsAg coding sequence of HBV DNA of certain Dane particlesof the ayw serotype can be isolated as a HhaI fragment. HBV DNA of Daneparticles of the same serotype may also exhibit different patterns ofrestriction sites.

Oligonucleotide probes can be devised based on the known sequences ofthe HBV genome and used to probe genomic or cDNA libraries for HBV genesencoding for the antigens useful in the present invention. The genes canthen be further isolated using standard techniques and, if desired,restriction enzymes employed to mutate the gene at desired portions ofthe full-length sequence. See, e.g., Sambrook et al., supra, for adescription of techniques used to obtain and isolate DNA.

Finally, the genes encoding the HBV antigens can be producedsynthetically, based on the known sequences. The nucleotide sequence canbe designed with the appropriate codons for the particular amino acidsequence desired. In general, one will select preferred codons for theintended host in which the sequence will be expressed. The completesequence is generally assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.,Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311.

Polynucleotides can comprise coding sequences for the variouspolypeptides which occur naturally or can include artificial sequenceswhich do not occur in nature. These polynucleotides can be ligated toform a coding sequence for a fusion protein, if desired, using standardmolecular biology techniques.

Once coding sequences have been prepared or isolated, such sequences canbe cloned into any suitable vector or replicon. Numerous cloning vectorsare known to those of skill in the art, and the selection of anappropriate cloning vector is a matter of choice. Suitable vectorsinclude, but are not limited to, plasmids, phages, transposons, cosmids,chromosomes or viruses which are capable of replication when associatedwith the proper control elements. The coding sequence is then placedunder the control of suitable control elements, depending on the systemto be used for expression. Thus, the coding sequence can be placed underthe control of a promoter, ribosome binding site (for bacterialexpression) and, optionally, an operator, so that the DNA sequence ofinterest is transcribed into RNA by a suitable transformant. The codingsequence may or may not contain a signal peptide or leader sequencewhich can later be removed by the host in post-translational processing.See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

If present, the signal sequence can be the native leader found inassociation with the HBV antigen of interest. Alternatively, aheterologous signal sequence can be present which can increase theefficiency of secretion. A number of representative leader sequences areknown in the art and include, without limitation, the yeast α-factorleader, the TPA signal peptide, the Ig signal peptide, and the like.Sequences for these and other leader sequences are well known in theart.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the sequencesrelative to the growth of the host cell. Regulatory sequences are knownto those of skill in the art, and examples include those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound.Other types of regulatory elements may also be present in the vector.For example, enhancer elements may be used herein to increase expressionlevels of the constructs. Examples include the SV40 early gene enhancer(Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman etal. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived fromhuman CMV (Boshart et al. (1985) Cell 41:521), such as elements includedin the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expressioncassette may further include an origin of replication for autonomousreplication in a suitable host cell, one or more selectable markers, oneor more restriction sites, a potential for high copy number and a strongpromoter.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe molecule of interest may be desirable to achieve this end. Forexample, in some cases it may be necessary to modify the sequence sothat it can be attached to the control sequences in the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

Any suitable expression vector can be constructed or utilized to expressany form of HBsAg of the invention. An exemplary vector is pCMVII, apUC19-based cloning vector designed for expression in mammalian cells.pCMVII comprises the following elements: human CMV IE enhancer/promoter,human CMV intron A, a human tissue plasminogen activator (tPA) leader, abovine growth hormone poly A terminator (BGHt), a ColE1 origin ofreplication, and an Amp R ampicillin resistance gene. For example,pCMVII-pS2-sAg can be used for expression of preS2-sAg. In this vector,the coding sequences for the sAg and preS2 domains of HBsAg have beeninserted into pCMVII between CMV intron A and BGHt. This vector can alsobe modified by, e.g., removing the preS2 domain or adding the codingsequence for the preS1 domain. These vectors are provided by way ofexample and are not intended to limit the scope of the invention. Theabove vectors are described in detail in U.S. Pat. No. 6,740,323,incorporated herein by reference in its entirety.

As explained above, it may also be desirable to produce mutants oranalogs of the polypeptide of interest. Mutants or analogs of HBVpolypeptides for use in the subject compositions may be prepared by thedeletion of a portion of the sequence encoding the molecule of interest,by insertion of a sequence, and/or by substitution of one or morenucleotides within the sequence. Techniques for modifying nucleotidesequences, such as site-directed mutagenesis, and the like, are wellknown to those skilled in the art. See, e.g., Sambrook et al., supra;Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448;Geisselsoder et al. (1987) BioTechniques 5:786; Zoller and Smith (1983)Methods Enzymol. 100:468; Dalbie-McFarland et al. (1982) Proc. Natl.Acad. Sci USA 79:6409.

The molecules can be expressed in a wide variety of systems, includinginsect, mammalian, bacterial, viral and yeast expression systems, allwell known in the art. For example, insect cell expression systems, suchas baculovirus systems, are known to those of skill in the art anddescribed in, e.g., Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987). Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).Similarly, bacterial and mammalian cell expression systems are wellknown in the art and described in, e.g., Sambrook et al., supra. Yeastexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

A number of appropriate host cells for use with the above systems arealso known. For example, mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human embryonic kidney cells, human hepatocellularcarcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”)cells, as well as others. Similarly, bacterial hosts such as E. coli,Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest canbe stably integrated into a host cell genome or maintained on a stableepisomal element in a suitable host cell using various gene deliverytechniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules areproduced by growing host cells transformed by an expression vectordescribed above under conditions whereby the protein is expressed. Theexpressed protein is then isolated from the host cells and purified. Ifthe expression system secretes the protein into growth media, theproduct can be purified directly from the media. If it is not secreted,it can be isolated from cell lysates. The selection of the appropriategrowth conditions and recovery methods are within the skill of the art.

The HBV antigens can also be synthesized using chemical polymersyntheses such as solid phase peptide synthesis. Such methods are knownto those skilled in the art. See, e.g., J. M. Stewart and J. D. Young,Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford,Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis,Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, AcademicPress, New York, (1980), pp. 3-254, for solid phase peptide synthesistechniques.

The HBV antigens, obtained as described above, are then used to producerabbit monoclonal antibodies for use in diagnostics.

Anti-HBV Antibodies

The HBV antigens can be used to produce HBV-specific polyclonal andmonoclonal antibodies for use in diagnostic and detection assays.HBV-specific polyclonal and monoclonal antibodies specifically bind toHBV antigens. In particular, the HBV antigens can be used to producepolyclonal antibodies by administering the HBV antigen to a mammal, suchas a mouse, a rat, a rabbit, a goat, or a horse. Serum from theimmunized animal is collected and the antibodies are purified from theplasma by, for example, precipitation with ammonium sulfate, followed bychromatography, preferably affinity chromatography. Techniques forproducing and processing polyclonal antisera are known in the art.

Rabbit and mouse monoclonal antibodies directed against HBV-specificepitopes present in the proteins can also be readily produced. In orderto produce such monoclonal antibodies, the mammal of interest, such as arabbit or mouse, is immunized, such as by mixing or emulsifying theantigen in saline, preferably in an adjuvant such as Freund's completeadjuvant (“FCA”), and injecting the mixture or emulsion parenterally(generally subcutaneously or intramuscularly). The animal is generallyboosted 2-6 weeks later with one or more injections of the antigen insaline, preferably using Freund's incomplete adjuvant (“FIA”). In oneembodiment, the animal is immunized with one or more HBsAg mutants,preferably a mixture of 2 to 5 different HBsAg mutants is used.Wild-type HBsAgs may also be included in the immunogen. In a preferredregime, the animal, preferably a rabbit, is initially immunized with awild type HBsAg, and thereafter boosted with one or more HBsAg mutants.Particularly useful as immunogen are HBsAg mutants which have been foundto occur naturally, e.g., D3, D2, D1, Y1, Y2, described further below.Antibodies may also be generated by in vitro immunization, using methodsknown in the art. See, e.g., James et al., J. Immunol. Meth. (1987)100:5-40.

Polyclonal antisera is then obtained from the immunized animal. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells (splenocytes) may be screened(after removal of nonspecifically adherent cells) by applying a cellsuspension to a plate or well coated with the antigen. B-cells,expressing membrane-bound immunoglobulin specific for the antigen, willbind to the plate, and are not rinsed away with the rest of thesuspension. Resulting B-cells, or all dissociated splenocytes, are theninduced to fuse with cells from an immortalized cell line (also termed a“fusion partner”), to form hybridomas. Typically, the fusion partnerincludes a property that allows selection of the resulting hybridomasusing specific media. For example, fusion partners can behypoxanthine/aminopterin/thymidine (HAT)-sensitive.

If rabbit-rabbit hybridomas are desired, the immortalized cell line willbe from a rabbit. Such rabbit-derived fusion partners are known in theart and include, for example, cells of lymphoid origin, such as cellsfrom a rabbit plasmacytoma as described in Spieker-Polet et al., Proc.Natl. Acad. Sci. USA (1995) 92:9348-9352 and U.S. Pat. No. 5,675,063, orthe TP-3 fusion partner described in U.S. Pat. No. 4,859,595,incorporated herein by reference in their entireties. If a rabbit-mousehybridoma or a rat-mouse or mouse-mouse hybridoma, or the like, isdesired, the mouse fusion partner will be derived from an immortalizedcell line from a mouse, such as a cell of lymphoid origin, typicallyfrom a mouse myeloma cell line. A number of such cell lines are known inthe art and are available from the ATCC.

Fusion is accomplished using techniques well known in the art. Chemicalsthat promote fusion are commonly referred to as fusogens. These agentsare extremely hydrophilic and facilitate membrane contact. Oneparticularly preferred method of cell fusion uses polyethylene glycol(PEG). Another method of cell fusion is electrofusion. In this method,cells are exposed to a predetermined electrical discharge that altersthe cell membrane potential. Additional methods for cell fusion includebridged-fusion methods. In this method, the antigen is biotinylated andthe fusion partner is avidinylated. When the cells are added together,an antigen-reactive B cell-antigen-biotin-avidin-fusion partner bridgeis formed. This permits the specific fusion of an antigen-reactive cellwith an immortalizing cell. The method may additionally employ chemicalor electrical means to facilitate cell fusion.

Following fusion, the cells are cultured in a selective medium (e.g.,HAT medium). In order to enhance antibody secretion, an agent that hassecretory stimulating effects can optionally be used, such as IL-6. See,e.g., Liguori et al., Hybridoma (2001) 20:189-198. The resultinghybridomas can be plated by limiting dilution, and are assayed for theproduction of antibodies which bind specifically to the immunizingantigen (and which do not bind to unrelated antigens). The selectedmonoclonal antibody-secreting hybridomas are then cultured either invitro (e.g., in tissue culture bottles or hollow fiber reactors), or invivo (e.g., as ascites in mice). For example, hybridomas producingHBV-specific antibodies can be identified using RIA or ELISA andisolated by cloning in semi-solid agar or by limiting dilution. Clonesproducing HBV-specific antibodies can isolated by another round ofscreening.

An alternative technique for generating the rabbit monoclonal antibodiesof the present invention is the selected lymphocyte antibody method(SLAM). This method involves identifying a single lymphocyte that isproducing an antibody with the desired specificity or function within alarge population of lymphoid cells. The genetic information that encodesthe specificity of the antibody (i.e., the immunoglobulin V_(H) andV_(L) DNA) is then rescued and cloned. See, e.g., Babcook et al., Proc.Natl. Acad. Sci. USA (1996) 93:7843-7848, for a description of thismethod.

For further descriptions of rabbit monoclonal antibodies and methods ofmaking the same from rabbit-rabbit and rabbit-mouse fusions, see, e.g.,U.S. Pat. Nos. 5,675,063 (rabbit-rabbit); 4,859,595 (rabbit-rabbit);5,472,868 (rabbit-mouse); and 4,977,081 (rabbit-mouse). For adescription of the production of conventional mouse monoclonalantibodies, see, e,g., Kohler and Milstein, Nature (1975) 256:495-497.

It may be desirable to provide chimeric antibodies. Chimeric antibodiescomposed of human and non-human amino acid sequences may be formed fromthe monoclonal antibody molecules described above to reduce theirimmunogenicity in humans (Winter et al. (1991) Nature 349:293; Lobuglioet al. (1989) Proc. Nat. Acad. Sci. USA 86:4220; Shaw et al. (1987) JImmunol. 138:4534; and Brown et al. (1987) Cancer Res. 47:3577;Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science239:1534; and Jones et al. (1986) Nature 321:522; EP Publication No.519,596, published 23 Dec. 1992; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994).

Antibody molecule fragments, e.g., F(ab′)₂, Fv, and sFv molecules, thatare capable of exhibiting immunological binding properties of the parentmonoclonal antibody molecule can be produced using known techniques.Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659; Hochman et al.(1976) Biochem 15:2706; Ehrlich et al. (1980) Biochem 19:4091; Huston etal. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879; and U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and 4,946,778, to Ladner etal.

In the alternative, a phage-display system can be used to expandmonoclonal antibody molecule populations in vitro. Saiki, et al. (1986)Nature 324:163; Scharf et al. (1986) Science 233:1076; U.S. Pat. Nos.4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392; Barbas,III et al. (1995) Methods: Comp. Meth Enzymol 8:94; Barbas, III et al.(1991) Proc Natl Acad Sci USA 88:7978.

Once generated, the phage display library can be used to improve theimmunological binding affinity of the Fab molecules using knowntechniques. See, e.g., Figini et al. (1994) J. Mol. Biol. 239:68. Thecoding sequences for the heavy and light chain portions of the Fabmolecules selected from the phage display library can be isolated orsynthesized, and cloned into any suitable vector or replicon forexpression. Any suitable expression system can be used, including thosedescribed above.

Polynucleotide sequences encoding the rabbit monoclonal antibodies andimmunoreactive fragments thereof, described above, are readily obtainedusing standard techniques, well known in the art, such as thosetechniques described above with respect to the HBsAgs.

Antibodies which are directed against HBV epitopes, are particularlyuseful for detecting the presence of HBV or HBV antigens in a sample,such as a serum sample from an HBV-infected human. An immunoassay for anHBV antigen may utilize one antibody or several antibodies either aloneor in combination with HBV antigens. An immunoassay for an HBV antigenmay use, for example, a monoclonal antibody directed towards an HBVepitope, a combination of monoclonal antibodies directed towardsepitopes of one HBV polypeptide, monoclonal antibodies directed towardsepitopes of different HBV polypeptides, polyclonal antibodies directedtowards the same HBV antigen, polyclonal antibodies directed towardsdifferent HBV antigens, or a combination of monoclonal and polyclonalantibodies. For example, both rabbit and mouse monoclonal antibodies canbe used in the subject assays. Immunoassay protocols may be based, forexample, upon competition, direct reaction, or sandwich type assaysusing, for example, labeled antibody and are described further below.The labels may be, for example, fluorescent, chemiluminescent, orradioactive.

The anti-HBV antibodies may further be used to isolate HBV particles orantigens by immunoaffinity columns. The antibodies can be affixed to asolid support by, for example, adsorption or by covalent linkage so thatthe antibodies retain their immunoselective activity. Optionally, spacergroups may be included so that the antigen binding site of the antibodyremains accessible. The immobilized antibodies can then be used to bindHBV particles or antigens from a biological sample, such as blood orplasma. The bound HBV particles or antigens are recovered from thecolumn matrix by, for example, a change in pH.

Preferred anti-HBV antibodies are those produced by the hybridoma celllines 99S9 (CMCC #12336) and 99S6 (CMCC #12337) (ATCC Accession Nos.PTA-6014 and PTA-6015, respectively) as well as antibody fragments (e.g.Fab, F(ab′)2, Fv, sFv) and chimeric or humanized antibodies derivedthereform.

HBV Diagnostic Assays

As explained above, the anti-HBV antibodies produced as described above,can be used in assays to identify HBV infection. The anti-HBV antibodiescan be used as either the capture component and/or the detectioncomponent in the assays, as described further below. Thus, the presenceof HBV in a biological sample can be determined by the presence of HBVantigens and/or anti-HBV antibodies as an indicator of HBV in thesample. The monoclonal antibodies can be used for detecting HBV in bloodsamples, including without limitation, in whole blood, serum, platelets,and plasma. The antibodies can be used to detect HBV infection in asubject, such as a human subject, as well as to detect HBV contaminationin donated blood samples by detecting the presence of HBV antigens,particularly HBsAgs, and HBV antibodies, depending on the assay used.Thus, aliquots from individual donated samples or pooled samples can bescreened for the presence of HBV and those samples or pooled samplescontaminated with HBV can be eliminated before they are combined. Inthis way, a blood supply substantially free of HBV contamination can beprovided. By “substantially free of HBV” is meant that the presence ofHBV is not detected using the assays described herein. Similarly, themethods of the present invention can be used to screen potential tissueand organ samples for transplantation and contaminated tissues andorgans can be discarded.

Assays for use herein include Western blots; agglutination tests;enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidinand biotin-streptavidin type assays; protein A- or protein G-mediatedimmunoassays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, strip immunoblot assays, and the like. Thereactions generally include detectable labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between the HBVantigen present in the sample and antibody or antibodies contactedtherewith.

The aforementioned assays generally involve separation of unboundantibodies or antigen in a liquid phase from a solid phase support towhich antigen-antibody complexes are bound. Solid supports which can beused in the practice of the invention include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

In one aspect of the invention, the anti-HBsAg antibodies, such as therabbit monoclonal antibodies described herein, are used for capture ordetection or both of HBV antigens, particularly HBsAgs, in a sample.Antibodies to the HBsAgs, produced as described above, can be used forthe capture or detection or both of HBV antigens in a sample. By“capture” of an analyte ( here HBV antigens in a sample) is meant thatthe analyte can be separated from other components of the sample byvirtue of the binding of the capture molecule. Typically, the capturemolecule is associated with a solid support, either directly orindirectly. Typically, the detection molecule is associated with adetectable label, either directly or indirectly.

Typically, a solid support is first reacted with a solid phase component(e.g., one or more of the anti-HBV antibodies) under suitable bindingconditions such that the component is sufficiently immobilized to thesupport. Sometimes, immobilization to the support can be enhanced byfirst coupling to a protein with better binding properties. Suitablecoupling proteins include, but are not limited to, protein A or proteinG, macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Alternatively, a streptavidin- or avidin-coated solidsupport can be used to immobilize a biotinylated antibody. Othermolecules that can be used to bind the antibody to the support includepolysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and the like. Such molecules and methodsof coupling these molecules are well known to those of ordinary skill inthe art. See, e.g., Brinkley, M. A. Bioconjugate Chem. (1992) 3:2-13;Hashida et al., J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu andStaros, International J. of Peptide and Protein Res. (1987) 30:117-124.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing the analyte (e.g., HBVantigens) under suitable binding conditions. After washing to remove anynon-bound analyte, a secondary binder moiety can be added under suitablebinding conditions, wherein the secondary binder is capable ofassociating selectively with the bound ligand. The presence of thesecondary binder can then be detected using techniques well known in theart.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated, directly or indirectly, with the rabbitanti-HBV antibodies according to the present invention. Rabbit anti-HBVantibodies directed against one or more HBV mutants as described abovecan be used. Preferably the rabbit monoclonal antibodies produced by thehybridoma line 99S9 or 99S6 are used. Additionally, other anti-HBVantibodies directed against wild-type HBsAgs can also be present, as canadditional mouse monoclonal antibodies directed against a wild-typeHBsAg or an HBsAg mutant. A biological sample containing or suspected ofcontaining HBV antigens is then added to the coated wells. After aperiod of incubation sufficient to allow antigen-antibody binding, theplate(s) can be washed to remove unbound moieties and a detectablylabeled secondary binding molecule added. The secondary binding moleculeis allowed to react with any captured sample, the plate washed and thepresence of the secondary binding molecule detected using methods wellknown in the art.

In one particular format, an ELISA antigen sandwich format is used. Inthis case, the solid support is coated with anti-HBV antibodies directedagainst one or more HBV mutants as described above. Anti-HBV antibodiesdirected against wild-type HBsAgs can also be present. The sample isthen contacted with the support under conditions that allow HBVantigens, if present, to bind one or more or the antibodies to form anantigen/antibody complex. Unbound antigens are removed and anenzyme-labeled antibody that reacts with the bound antigen/antibodycomplex, such as a labeled anti-HBsAg antibody, is added. An enzymesubstrate is used to generate a signal. In this particular embodiment,the anti-HBV antibodies that are coated on the solid support can berabbit monoclonal antibodies of the present invention, preferably theantibodies produced by hybridoma 99S9 or hybridoma 99S6, or both.Alternatively, or in addition, the detectably labeled antibody can be arabbit monoclonal antibody of the present invention, preferably theantibodies produced by hybridoma 99S9 or hybridoma 99S6, or both.

In another embodiment, the presence of bound HBV analytes from abiological sample can be readily detected using a secondary bindercomprising an antibody directed against the antigen ligands. A number ofanti-human immunoglobulin (Ig) molecules are known in the art which canbe readily conjugated to a detectable enzyme label, such as horseradishperoxidase, alkaline phosphatase or urease, using methods known to thoseof skill in the art. An appropriate enzyme substrate is then used togenerate a detectable signal. In other related embodiments,competitive-type ELISA techniques can be practiced using methods knownto those skilled in the art.

The rabbit anti-HBV antibodies of the invention can also be used in anindirect ELISA, for example, an indirect IgG ELISA, as follows. Theantibodies specific for HBV surface antigens are attached to a solidsupport. Protein A or protein G can be used to immobilize the antibodieson the solid support. The support is then contacted with HBsAg underconditions that allow binding to the anti-HBV antibodies bound to thesupport to form antibody/antigen complexes. Unbound antigens are removedand the support is contacted with a sample to be tested for the presenceof human IgG to HBV under conditions that allow binding of humananti-HBV IgG, if present, to the antigens in the antibody/antigencomplexes. The presence of bound anti-HBV IgG can be detected using adetectably labeled anti-human IgG antibody. In like manner, the presenceof human IgM to HBV can be detected by using labeled anti-human IgM tobind to the antibody/antigen complexes.

The rabbit anti-HBV antibodies of the invention can also be used in acapture ELISA, for example, an IgM capture ELISA, as follows. Anti-humanIgM antibodies (e.g., goat anti-human IgM antibodies) are attached to asolid support, the support is contacted with a sample to be tested forthe presence of human IgM to HBV, under conditions that would allow thebinding of the anti-HBV IgM, if present, to one or more of theanti-human IgM antibodies attached to the solid support, to formantibody/antibody complexes. The HBsAgs (e.g., mutant and/or wild-type)are added under conditions that would allow binding to the anti-HBV IgMin the antibody/antibody complexes forming an antibody/antibody/antigencomplex. Unbound antigens are removed and detectably labeled anti-HBVantibodies, produced as described above, are added under conditions thatallow binding to the bound antigens. The presence of IgM to HBV in thesample is determined by the presence of the detectably labeled anti-HBVantibodies to the bound anti-human IgM Ab/human anti-HBV IgM/antigencomplexes attached to the solid support.

While some of the foregoing assay formats are termed “ELISA” (EnzymeLinked ImmunoSorbant Assay) assays, it will be apparent to one of skillin the art that the use of a detectable label other than an “enzymelinked” binding moiety is possible and may be desirable in manysituations. Other suitable detectable labels are described herein andare well known in the art.

Assays can also be conducted in solution, such that the HBV antigens orantibodies and ligands specific for these molecules form complexes underprecipitating conditions. In one particular embodiment, the moleculescan be attached to a solid phase particle (e.g., an agarose bead or thelike) using coupling techniques known in the art, such as by directchemical or indirect coupling. The coated particle is then contactedunder suitable binding conditions with a biological sample suspected ofcontaining HBV antibodies or antigens. Cross-linking between boundantibodies causes the formation of complex aggregates which can beprecipitated and separated from the sample using washing and/orcentrifugation. The reaction mixture can be analyzed to determine thepresence or absence of complexes using any of a number of standardmethods, such as those immunodiagnostic methods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided,wherein, for example, a polyclonal population of antibodies from abiological sample suspected of containing HBV antibodies is immobilizedto a substrate. An initial affinity purification of the sample can becarried out using immobilized antigens. The resultant sample preparationwill thus only contain anti-HBV moieties, avoiding potential nonspecificbinding properties in the affinity support. A number of methods ofimmobilizing immunoglobulins (either intact or in specific fragments) athigh yield and good retention of antigen binding activity are known inthe art. For example, protein A or protein G can be used to immobilizeimmunoglobulin molecules to the solid support. Once the immunoglobulinmolecules have been immobilized to provide an immunoaffinity matrix, HBVantigens, such as HBsAgs, are contacted with the bound antibodies undersuitable binding conditions. After any non-specifically bound HBVantigen has been washed from the immunoaffinity support, the presence ofbound antigen can be determined by assaying for label using methodsknown in the art. For example, an enzymatically labeled antibody thatreacts with the bound antigen/antibody complex, such as a labeledanti-HBsAg antibody, produced as described above, is added. An enzymesubstrate is used to generate a signal.

In another embodiment of the invention, a strip immunoblot assay (SIA)is used to detect HBV antigens in a biological sample. For example, oneor more of the rabbit monoclonal antibodies described above, andoptionally mouse monoclonal antibodies directed against an HBsAg, can beimmobilized on the test strip as capture reagents. SIA techniques arewell known in the art and combine traditional western and dot blottingtechniques, e.g., the RIBA® (Chiron Corp., Emeryville, Calif.) SIA. Inthese assays, the antibodies are immobilized as individual, discreteportions, e.g., as bands or dots, on a membranous support, or may beimmobilized as a mixture in a single portion. Thus, by “discretelyimmobilized” on a membrane support is meant that the antibodies arepresent as separate components and not mixed, such that reactivity orlack thereof with each of the capture reagents present can be assessed.A biological sample suspected of containing HBV antigens is then reactedwith the test membrane. Visualization of reactivity in the biologicalsample can be accomplished using anti-HBV antibody enzyme-conjugates inconjunction with a colorimetric enzyme substrate. Alternatively, therabbit monoclonal antibodies described above can be used forvisualization of the bound antibody-antigen complexes. The test stripfor this alternative embodiment may be prepared using, e.g., mousemonoclonal antibodies directed against HBsAg. The assay can be performedmanually or used in an automated format.

Solid supports which can be used in the practice of the strip immunoblotassays include, but are not limited to, membrane supports derived from anumber of primary polymers including cellulose, polyamide (nylon),polyacrylonitrile, polyvinylidene difluoride, polysulfone,polypropylene, polyester, polyethylene and composite resins consistingof combinations or derivatives of the above. Particularly preferred aresupports derived from cellulose, such as nitrocellulose membranes, aswell as nylon membranes. The substrate generally includes the desiredmembrane with an inert plastic backing as a support.

The above-described assay reagents, including the rabbit monoclonalantibodies and/or the HBsAgs described herein, the solid supports withbound reagents, as well as other detection reagents, can be provided inkits, with suitable instructions and other necessary reagents, in orderto conduct the assays as described above. The kit may also includecontrol formulations (positive and/or negative), labeled reagents whenthe assay format requires same and signal generating reagents (e.g.,enzyme substrate) if the label does not generate a signal directly.Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying outthe assay usually will be included in the kit. The kit can also contain,depending on the particular assay used, other packaged reagents andmaterials (i.e. wash buffers and the like). Standard assays, such asthose described above, can be conducted using these kits.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of Rabbit Monoclonal Antibodies

To produce monoclonal antibodies that confer sufficientimmunoreactivities to the various mutants of interest as well as to thewild-type HBsAg, three white female adult New Zealand rabbits wereimmunized with wild-type HBsAg (adw) and (ayw) antigens (shown in FIGS.2A and 2B, respectively), followed by boosts every two weeks with acocktail of the wild-type antigens and five major HBV recombinant mutantantigens, designated D1 (having an A for F substitution at amino acidposition 134), D2 (having an S for F substitution at amino acid position134), D3 (having an R for G substitution at amino acid 145), Y1 (havingan L for S substitution at amino acid 143, and Y2 (having an R for Qsubstitution at amino acid 129 and a T for M substitution at amino acid133).

The immune B cells from the spleen of the most immunoreactive rabbitwere fused to rabbit plasmacytoma cells to produce hybridomas,essentially as described in Spieker-Polet et al., Proc. Natl. Acad. Sci.USA (1995) 92:9348-9352. Briefly, 1.5-3×10 ⁸ lymphocytes from animmunized rabbit were fused with a fusion partner derived from a rabbitplasmacytoma cell line (for example, 240E 1-1-2, described in U.S. Pat.No. 5,675,063, incorporated herein by reference in its entirety;Spieker-Polet et al., Proc. Natl. Acad. Sci. USA (1995) 92:9348-9352) ata ratio of 2:1 with 50% PEG 4000 at 37 C in serum-free medium. The cellswere distributed in 96-well cell culture plates at approximately 1×10⁵lymphocytes per well, in medium with 15% FBS (or FCS). After 48 hr, HATmedium was added. Medium was changed 2-3 times before screening.Usually, hybridoma colonies were ready for screening in 3-5 weeks.Supernatants were tested for the presence of antibody specific for theimmunogen, by ELISA. Immunohistochemistry was used as a secondaryscreening assay. The hybridomas were sub-cloned by limit dilution. Forfeeder cells, the fusion partner at 2×10⁴ cells per well was used.

A total of 3000 clones were screened, and 38 clones (Table 1) wereidentified as potential candidates for further study. These clones werethen subcloned and tested for the production of antibodies with the bestreactivity against all seven HBV surface antigens used for the antigencocktail, both mutant and wild-types (see Table 1). The resultsindicated that of the 38 clones evaluated, 4 clones (as highlighted inTable 1) had the broadest immunoreactivities to HBsAg mutants. Thus,these 4 HBV rabbit hybridomas were scaled up, purified and furtherevaluated.

After extensive analyses of the selected 38 HBV hybridoma clones, twoclones, 99S9 (ATCC Accession No. PTA-6014) and 99S6 (ATCC Accession No.PTA 6015), were demonstrated to produce antibodies with very broadimmunoreactivities against the HBV surface Ag mutant panel. Inparticular, an ELISA was used to compare the immunoreactivity of twomouse monoclonal antibodies, mMAb1 and mMAb2 (mouse monoclonalantibodies directed against HBsAg) and the rabbit monoclonal antibodiesproduced by the 99S9 and 99S6 hybridoma cell lines with the variousmutant HBsAgs described above. Additionally, the mouse monoclonalantibodies and rabbit monoclonal antibodies from hybridomas 99S6 and99S9 were tested using a BIACORE 3000 system (Biacore AB, Piscataway,N.J.). This system provides real-time biomolecular interaction analysis(BIA) using surface plasmon resonance (SPR) technology. SPR-basedbiosensors monitor interactions by measuring the mass concentration ofbiomolecules close to a surface. The surface is made specific byattaching one of the interacting partners. Sample containing the otherpartner(s) flows over the surface. When molecules from the sample bindto the interactant attached to the surface, the local concentrationchanges and an SPR response is measured. The response is directlyproportional to the mass of molecules that bind to the surface. In thiscase, Goat anti-rabbit IgG antibody (to test immunoreactivity of therabbit monoclonal antibodies) or goat anti-mouse IgG (to testimmunoreactivity of the mouse monoclonal antibodies) was immobilized ona sensor chip to provide the surface-capturing antibodies. The rabbit ormouse monoclonal antibodies described above (ligands) were then capturedby the surface capturing antibodies. The mutant and wild-type HBsAgs(analytes) were then passed through this surface and theantibody/antigen interactions were detected and measured using a BIACORESPR optical device.

FIGS. 3A-3D and Table 2 summarize the BIACORE analysis results of rabbitmonoclonal antibodies from 99S6 (FIG. 3B) and 99S9 (FIG. 3D), incomparison with the mouse antibodies mMAb1 (FIG. 3C) and mMAb2 (FIG.3A). For these and the following studies, a number of different mousemonoclonal antibodies to HBsAg were used (mMAb1, mMAb2, mMAb3, mMAb4,mMAb5, and mMAb6). Several commercially available mouse monoclonalantibodies were also used. As shown in FIGS. 3A-3D, while mousemonoclonal antibody mMAb2 was deficient in binding to the mutant D3, andmMAb1 was deficient in binding to mutants D3 and Y1, both rabbithybridoma clones 99S9 and 99S6 produced antibodies with significantbinding activities to all 7 antigens, including to D3 and Y1. Inaddition, the BIACORE results also evidenced that binding of rabbitmonoclonal antibodies (from hybridoma clones 99S9 and 99S6) to themutants D3 and Y1 was very stable (FIG. 3). These results demonstratedthat the rabbit monoclonal antibodies from clones 99S9 and 99S6 had muchstronger immunoreactivities against mutant antigens D3 and Y1 whileretaining immunoreactivities for the other antigens, and thusdemonstrated that the rabbit antibodies had much broaderimmunoreactivities for the various HBsAg mutants compared to the twomouse monoclonal antibodies.

To further demonstrate the advantages of the rabbit monoclonalantibodies for HBV variant (i.e., mutant) detection, the rabbitmonoclonal antibody from 99S9 was tested in comparison with the mousemonoclonal antibodies as the capture or as the detection antibody insandwich ELISA assays for detection of the wild-type and variants ofHBsAg. In these experiments, two sets of ELISA plates were coated witheither single rabbit monoclonal antibody (99S9) or a cocktail of twoanti-HBsAg mouse monoclonal antibodies, mMAb2 and 160S11 (BD BiosciencesPharmingen (San Diego, Calif.). These two sets of capture plates werepaired with a single 99S9 horseradish peroxidase (HRP) detection system,or paired with a mouse monoclonal antibody cocktail containing a mixtureof 4 HRP-labeled anti-HBsAg MAbs (mMAb1, M01077 (Fitzgerald IndustriesInternational, Concord, Mass.), M01079 (Fitzgerald IndustriesInternational, Concord, Mass.), and mMAb3).

The results were consistent with the results from the BIACORE analysisstudy. The rabbit monoclonal antibody from 99S9 had broaderimmunoreactivities compared with the mouse monoclonal antibodies. When99S9 was tested as the sole capture and detection antibody (99S9 as thecapture antibody and HRP-99S9 as the detection antibody), it was capableof detecting all 7 antigens, while the mouse monoclonal antibodycocktails (mMAb2/160S11 as the capture antibody andHRP-mMAb1/M77/M79/2D11 as the detection antibody) failed to detect D3antigen (Table 3). These results showed that a single rabbit monoclonalantibody could sufficiently replace the multiple mouse monoclonalantibodies in ELISA assays used for detection of some major escapedmutants.

To summarize the above experiments, rabbit monoclonal antibody 99S9 hadaffinity to 7 antigens tested and showed much slower off-rates for allof them, especially for D3 and Y1 (FIG. 3 and Table 2). Although themouse monoclonal mMAb1 seemed to have significantly higher affinity formutant D1, D2, and wild-types adw and ayw, it was incapable of bindingto mutant antigens D3 and Y1 (FIG. 3 and Table 2). The mouse monoclonalantibody mMAb2 had overall lower affinities for the most HBV mutantantigens and was incapable of binding to mutant antigen D3 (FIG. 3 andTable 2). The rabbit monoclonal antibody 99S9 alone was sufficient toreplace the combinations of multiple mouse monoclonal antibodies formore effective capture and/or detection of mutant D3 (Table 3).

To test the ability of the rabbit monoclonal antibodies to detect otherHBsAg variants, additional ELISAs were carried out using either onlymouse monoclonal antibodies against HBsAg (mMAb ELISA) or a combinationof mouse monoclonals and rabbit monoclonals against HBsAg (rMAb ELISA).For the mMAb ELISA, 5 different anti-HBsAg mouse monoclonal antibodieswere used, 2 for capture (mMAb2 and mMAb4) and 3 for detection (mMAb1,mMAb5 and mMAb6). For the rMAb ELISA, 2 of the mouse monoclonalantibodies used for detection (mMAb5 and mMAb6) were replaced by asingle rabbit monoclonal antibody (99S9). The capture antibodies werebiotinylated and immobilized on streptavidin-coated wells. The detectionantibodies were conjugated with horseradish peroxidase (HRP). Table 4shows the results with a number of HBsAg variants. The rMAb ELISAdetected all of the variants that were detected in the mMAb ELISA and,additionally, detected 2 variants that were not detected in the mMAbELISA, the P120Q variant and the P142S variant. Thus, the ELISA usingthe rabbit monoclonal antibody (99S9) was able to detect more HBsAgvariants using fewer antibodies than the ELISA using only the mousemonoclonal antibodies.

Therefore, rabbit monoclonal antibodies provide a powerful tool forbetter detection of HBV variant antigens.

Thus, novel monoclonal antibodies and methods for detecting HBVinfection are disclosed. Although preferred embodiments of the subjectinvention have been described in some detail, it is understood thatobvious variations can be made without departing from the spirit and thescope of the invention as described herein. TABLE 1 HBV Mutant AntigensHBV Wild-type Antigens ID D1 D2 D3 Y1 Y2 ADW AYW 27S-2 2.95 2.96 2.750.70 1.06 3.30 0.75 64S1 0.14 0.13 0.07 0.00 0.00 0.10 0.00 64S2 0.000.00 0.00 0.00 0.00 0.00 0.00 64S3 0.05 0.04 0.01 0.00 0.00 0.02 0.0064S4 0.00 0.00 0.00 0.01 0.00 0.00 0.00 71S1 2.24 1.90 1.40 0.05 0.061.70 0.07 71S4 3.07 2.90 2.70 0.21 0.22 2.80 0.24 71S5 3.38 3.40 3.200.29 0.35 3.00 0.35 71S7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 71S8 0.000.00 0.00 0.00 0.00 0.00 0.00 71S12 0.00 0.00 0.00 0.00 0.00 0.00 0.0071S14 3.06 0.00 3.20 0.32 0.32 3.00 0.34 71S15 2.93 3.10 3.00 0.28 0.262.70 0.30 71S16 3.13 3.00 2.98 0.32 0.35 3.30 0.40 71S17 3.33 3.30 2.770.38 0.48 3.00 0.53 96S1 3.45 3.40 2.87 3.10 3.30 2.70 2.92 99S1 3.372.80 2.90 1.40 1.50 2.90 1.70 99S4 2.78 2.70 2.40 0.73 0.80 3.00 1.2099S6 3.01 3.20 2.60 1.05 1.20 3.30 1.45 99S7 2.21 1.99 1.80 0.40 0.542.40 0.78 99S8 2.58 2.30 2.30 0.60 0.66 2.70 0.90 99S9 3.62 2.80 2.801.40 1.40 2.80 1.68 99S10 2.50 2.40 2.30 0.80 0.90 2.50 1.06 116S1 3.203.30 0.00 2.50 2.40 2.30 2.80 116S2 2.30 1.90 0.01 1.70 1.60 2.44 1.99116S3 2.70 2.60 0.00 2.40 1.98 2.60 2.70 116S4 2.80 2.60 0.00 2.20 1.872.80 2.45 116S5 1.90 1.70 0.00 1.60 1.30 2.01 2.03 121S2 3.07 2.96 2.900.33 0.00 3.07 2.40 121S4 3.70 3.10 2.90 0.36 0.00 3.20 2.60 121S5 3.012.70 2.90 0.31 0.00 2.80 2.10 121S6 3.70 3.40 2.80 0.42 0.00 3.10 2.60121S8 2.80 3.00 3.00 1.48 0.10 2.90 2.70 121S9 3.20 3.20 2.90 0.27 0.062.90 1.80 123S3 1.65 2.10 0.01 0.00 1.70 0.00 0.00 123S4 1.45 1.70 0.010.00 1.40 0.00 0.00 123S8 1.00 1.30 0.00 0.02 1.10 0.00 0.00 123S12 1.051.40 0.00 0.02 1.10 0.00 0.00Capture: ELISA plates were coated with the 7 different antigens.Sample Size: 200 μl (1:100 dilution of cell culture in the specimendiluent).Detection: Goat anti rabbit (fab)′2-HRP conjugate.

TABLE 2 ELISA BIACORE HBV Mutation mMAb2/ Mouse mAb Rabbit mAbrecombinant Site mMAb1 (mMAb1) (mMAb2) (99S6) (99S9) D1 F134A ++ +++++ +++ + D2 F134S ++ +++++ ++ ++ + D3 G145R (−, +/−, −) − or +/− − + + Y1S143L (−, −) − + + + Y2 129Q/133T +++ no result no result no result noresult HBV Wild- Mutation mMAb2/ (mMAb1) (mMAb2) (99S6) (99S9) type SitemMAb1 adw +++ +++++ ++ + ++ ayw + +++++ + + +

TABLE 3 Capture: 2 Mouse MnAbs mMAb2/160S11 1 Rabbit MnAb99S9 Detection4 Mouse MnAbs 4 Mouse MnAbs mMAb1/M77/M79/ 1 Rabbit MnAb mMAb1/M77/M79/1 Rabbit MnAb mMAb3 99S9-HRP mMAb3 99S9-HRP Testing samples OD OD OD ODAdw (3 ng/test) 2.84 0.73 2.45 0.53 Ayw (3 ng/test) 2.64 0.31 2.67 0.31D1 (3 ng/test) 2.35 0.56 2.02 0.37 D2 (3 ng/test) 1.90 0.56 1.69 0.46 D3(3 ng/test) 0.02 0.08 0.01 0.05 Y1 (3 ng/test) 0.87 0.30 0.81 0.21 Y2 (3ng/test) 1.69 0.32 1.25 0.20 P120Q (A)(3 ng/test) 1.65 0.17 1.00 0.07Cut Off 0.07 0.09 0.01 0.03 Testing samples S/C S/C S/C S/C adw (3ng/test) 40.6 8.1 244.5 17.8 ayw (3 ng/test) 38.2 3.5 266.7 10.2 D1 (3ng/test) 33.6 6.2 201.9 12.2 D2 (3 ng/test) 27.2 6.2 168.8 15.2 D3 (3ng/test) 0.3 0.9 0.9 1.8 Y1 (3 ng/test) 12.4 3.3 80.6 7.0 Y2 (3 ng/test)24.1 3.5 125.3 6.6 P120Q (A)(3 ng/test) 23.6 1.9 99.7 2.3Capture 1: rabbit Monoclonal 99S9 Capture 2: Mouse Monoclonals mMAb2,160S11Detection 1: mMnAb-HRP (mMAb1, M01077, M01079, mMAb3) Detection 2:rabbit MnAb-HRP 99S9

TABLE 4 ALU Sample Mouse MAb Assay Rabbit MAb Assay Positive Control127.15 143.06 Negative Control 3.34 4.42 adw 0.5 ng/ml 327.62 334.99 ayw0.5 ng/ml 299.61 302.06 G145R 1 ng/ml 167.36 145.65 S143L 1 ng/ml 33.5939.67 P120Q 1 ng/ml 33.06 68.81 P142L 0.2 ng/ml 21.01 35.77 D144A 0.2ng/ml 48.40 37.54 F134S 0.2 ng/ml 85.83 88.22 F134A 0.2 ng/ml 140.45145.80 Y118K 1:9000 26.83 28.68 Y1188 1:9000 46.65 49.10 Y131A 1:900085.89 86.72 T126N (Neat) 156.28 166.39 Q129H (Neat) 222.93 291.02 M133D(Neat) 51.13 66.07 P142S (1:10 Dil) 41.48 223.58 D144N (1:600 Dil) 61.5581.05Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. The accession number indicated was assignedafter successful viability testing, and the requisite fees were paid.The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for a period ofthirty (30) years from the date of deposit and at least five (5) yearsafter the most recent request for the furnishing of a sample of thedeposit by the depository. The organisms will be made available by theATCC under the terms of the Budapest Treaty, which assures permanent andunrestricted availability of the cultures to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.12). Upon the granting of a patent, allrestrictions on the availability to the public of the deposited cultureswill be irrevocably removed.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The nucleic acid sequences of these plasmids, as well asthe amino acid sequences of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with the description herein. A license may be required to make,use, or sell the deposited materials, and no such license is herebygranted. Hybridoma Deposit Date ATCC No. 99S6 (CMCC #12337) May 26, 2004PTA-6015 99S9 (CMCC #12336) May 26, 2004 PTA-6014

1. A hepatitis B virus (HBV) rabbit monoclonal antibody that recognizesan HBsAg mutant with a mutation in the “a” determinant region, or animmunoreactive fragment thereof.
 2. The rabbit monoclonal antibody ofclaim 1, wherein the antibody recognizes more than one HBsAg mutant witha mutation in the “a” determinant region.
 3. The rabbit monoclonalantibody of claim 1, wherein the antibody also recognizes a wild-typeHBsAg.
 4. The rabbit monoclonal antibody of claim 1, wherein the HBsAgmutant is a mutant sAg.
 5. The rabbit monoclonal antibody of claim 4,wherein the HBsAg mutant comprises the sequence of the “a” determinantregion of F134A, F134S, G145R, S143L, P142S or Q129R/M133T.
 6. Therabbit monoclonal antibody of claim 5, wherein the antibody recognizesan HBsAg selected from the group consisting of F134A, F134S, G145R,S143L, P142S and Q129R/M133T.
 7. The rabbit monoclonal antibody of claim5, wherein the antibody recognizes at least two mutant HBsAg selectedfrom the group consisting of F134A, F134S, G145R, S143L, P142S andQ129R/M133T.
 8. The rabbit monoclonal antibody of claim 5, wherein theantibody recognizes the mutant HBsAgs F134A, F134S, G145R, S143L, P142Sand Q129R/M133T.
 9. The rabbit monoclonal antibody of any of claim 1,wherein said antibody is produced using a rabbit-rabbit hybridoma. 10.The rabbit monoclonal antibody of claim 1, wherein said antibody isproduced using a rabbit-mouse hybridoma.
 11. Hybridoma 99S6 (ATCCAccession number PTA-6015).
 12. Hybridoma 99S9 (ATCC Accession numberPTA-6014).
 13. An antibody produced by hybridoma 99S6 (ATCC Accessionnumber PTA-6015).
 14. An antibody produced by hybridoma 99S9 (ATCCAccession number PTA-6014).
 15. The fragment of claim 1, wherein thefragment is a Fab, F(ab′)₂, Fv or an sFv fragment.
 16. A rabbitmonoclonal antibody that recognizes the same epitope as an antibodyproduced by hybridoma 99S6 (ATTC Accession No. PTA-6015) or 99S9 (ATCCAccession No. PTA-6014).
 17. A method of detecting HBV surface antigensin a biological sample, comprising: (a) contacting said biologicalsample with at least one rabbit monoclonal antibody according to claim1, under conditions which allow HBV antigens, when present in thebiological sample, to bind to said antibody to form an antibody/antigencomplex; and (b) detecting the presence or absence of saidantibody/antigen complex, thereby detecting the presence or absence ofHBV surface antigens in said sample.
 18. The method of claim 17, whereinsaid at least one rabbit monoclonal antibody is detectably labeled. 19.The method of claim 17, wherein the method further comprises reactingsaid biological sample with one or more additional antibodies directedagainst a wild-type HBsAg or an HBsAg mutant with a mutation in the “a”determinant region.
 20. The method of claim 19, wherein the one or moreadditional antibodies comprise an additional monoclonal antibody. 21.The method of claim 20, wherein the one or more additional antibodiescomprise a mouse monoclonal antibody.
 22. An immunodiagnostic test kitfor detecting HBV infection, said test kit comprising: (a) at least onerabbit monoclonal antibody, or immunoreactive fragment thereof accordingto claim 1; and (b) instructions for conducting the immunodiagnostictest.
 23. The immunodiagnostic test kit of claim 22, wherein the testkit further comprises one or more additional antibodies directed againsta wild-type HBsAg or an HBsAg mutant with a mutation in the “a”determinant region.
 24. The immunodiagnostic test kit of claim 23,wherein the one or more additional antibodies comprise an additionalmonoclonal antibody.
 25. The immunodiagnostic test kit of claim 24,wherein the one or more additional antibodies comprise a mousemonoclonal antibody.
 26. A solid support comprising at least one rabbitmonoclonal antibody or immunoreactive fragment thereof according toclaim
 1. 27. The solid support of claim 26, wherein the support furthercomprises one or more additional antibodies directed against a wild-typeHBsAg or an HBsAg mutant with a mutation in the “a” determinant region.28. The solid support of claim 27, wherein the one or more additionalantibodies comprise an additional monoclonal antibody.
 29. The solidsupport of claim 28, wherein the one or more additional antibodiescomprise a mouse monoclonal antibody.
 30. The solid support of claim 26,further comprising at least two internal controls, wherein one of thecontrols defines the lower detection limit for a positive result in animmunoassay using the solid support and the other control defines ahighly positive result in an immunoassay using the solid support. 31.The solid support of claim 26, wherein the solid support is anitrocellulose strip.
 32. An immunodiagnostic test kit for detectingHBV, said test kit comprising: (a) a solid support according to claim26; and (b) instructions for conducting the immunodiagnostic test.
 33. Amethod of detecting the presence of HBV surface antigens in a biologicalsample, said method comprising: (a) providing a biological sample; (b)providing a solid support according to claim 26; (c) contacting saidbiological sample with said solid support, under conditions which allowHBV surface antigens, if present in the biological sample, to bind withat least one of the rabbit monoclonal antibodies to form anantibody/antigen complex; and (d) detecting the presence of theantibody/antigen complex, thereby detecting the presence of HBV surfaceantigens in the biological sample.
 34. The method of claim 33, furthercomprising: (e) removing unbound HBV antigens; (f) providing one or moremoieties capable of associating with said antibody/antigen complex; and(g) detecting the presence of said one or more moieties, therebydetecting the presence of HBV surface antigens in the biological sample.35. The method of claim 34, wherein said one or more moieties comprisesa detectably labeled HBV antibody.
 36. The method of claim 35, whereinsaid detectably labeled HBV antibody is a rabbit monoclonal antibodythat recognizes an HBsAg mutant with a mutation in the “a” determinantregion, or an immunoreactive fragment thereof.
 37. The method of claim35, wherein the detectable label is an enzyme.
 38. The method of claim17, wherein said biological sample is from a human blood sample.
 39. Amethod of detecting the presence of anti-HBsAg antibodies in abiological sample, said method comprising: (a) providing a solid supportaccording to claim 26; (b) contacting said solid support with one ormore HBsAgs, under conditions which allow the one or more HBsAgs to bindwith at least one of the rabbit monoclonal antibodies to form anantibody/antigen complex; (d) contacting said solid support having saidantibody/antigen complex with a biological sample, under conditionswhich allow anti-HBsAg antibodies, if present in the biological sample,to bind with said antibody/antigen complex to form anantibody/antigen/antibody complex; and (e) detecting the presence of theantibody/antigen/antibody complex, thereby detecting the presence ofanti-HBsAg antibodies in the biological sample.
 40. The method of claim39, further comprising: (f) removing unbound antibodies; (g) providingone or more moieties capable of associating with saidantibody/antigen/antibody complex; and (h) detecting the presence ofsaid one or more moieties, thereby detecting the presence of anti-HBsAgantibodies in the biological sample.
 41. The method of claim 40, whereinsaid one or more moieties comprises a detectably labeled immunoglobulinmolecule.
 42. A method of preparing a blood supply comprising wholeblood, platelets, plasma or serum, substantially free of HBV comprising:(a) screening aliquots of whole blood, platelets, plasma or serum fromcollected blood samples by the method of claim 38; (b) eliminating anysamples in which an HBV antigen is detected; and (c) combining samplesin which no HBV antigen is detected to provide a blood supplysubstantially free of HBV.
 43. A method of preparing a blood supplycomprising whole blood, platelets, plasma or serum, substantially freeof HBV comprising: (a) screening aliquots of whole blood, platelets,plasma or serum from collected blood samples by the method of claim 39;(b) eliminating any samples in which an anti-HBsAg antibody is detected;and (c) combining samples in which no anti-HBsAg antibody is detected toprovide a blood supply substantially free of HBV.
 44. A method ofscreening a donated tissue or organ prior to transplantation to providea tissue or organ substantially free of HBV comprising: (a) screening asample from said tissue or organ by the method of claim 17; (b)eliminating a tissue or organ in which an HBV antigen is detected toprovide a tissue or organ substantially free of HBV.
 45. A method ofscreening a donated tissue or organ prior to transplantation to providea tissue or organ substantially free of HBV comprising: (a) screening asample from said tissue or organ by the method of claim 39; (b)eliminating a tissue or organ in which an anti-HBsAg antibody isdetected to provide a tissue or organ substantially free of HBV.
 46. Amethod of preparing an anti-HBV rabbit monoclonal antibody, said methodcomprising: (a) immunizing a rabbit with an HBsAg mutant with a mutationin the “a” determinant region; (b) fusing cells that produce antibodiesagainst the HBsAg mutant from said rabbit with a cell from animmortalized cell line to produce a hybridoma; (c) selecting for saidhybridoma; (d) culturing said selected hybridoma; and (e) collecting theantibody secreted by said cultured hybridoma.
 47. The method of claim46, wherein said immunizing step comprises immunizing a rabbit with morethan one HBsAg mutant.
 48. The method of claim 46, wherein saidantibody-producing cells are rabbit splenocytes.
 49. The method of claim48, wherein said splenocytes are fused with a cell from an immortalizedrabbit cell line to produce a rabbit-rabbit hybridoma.
 50. The method ofclaim 49, wherein the immortalized rabbit cell line is a rabbitplasmacytoma.
 51. The method of claim 48, wherein said splenocytes arefused with a cell from an immortalized mouse cell line to produce arabbit-mouse hybridoma.
 52. The method of claim 46, wherein the HBsAgmutant is a mutant sAg.
 53. The method of claim 52, wherein the HBsAgmutant comprises the sequence of the “a” determinant region of F134A,F134S, G145R, S143L, P142S or Q129R/M133T.
 54. The method of claim 53,wherein the HBsAg mutant comprises F134A, F134S, G145R, S143L, P142S orQ129R/M133T.
 55. The method of claim 46, wherein the rabbit is immunizedwith at least two HBsAg mutants with different mutations in the “a”determinant region.
 56. The method of claim 53, wherein the rabbit isimmunized with at least two HBsAg mutants selected from the groupconsisting of F134A, F134S, G145R, S143L, P142S or Q129R/M133T.
 57. Themethod of claim 53, wherein the rabbit is immunized with HBsAg mutantsF134A, F134S, G145R, S143L, P142S and Q129R/M133T.
 58. The method ofclaim 46, wherein the rabbit is additionally immunized with a wild typeHBsAg.
 59. An anti-HBV rabbit monoclonal antibody produced by a methodaccording to claim
 46. 60. A method of preparing a rabbit-rabbithybridoma comprising: (a) immunizing a rabbit with an HBsAg mutant witha mutation in the “a” determinant region; (b) fusing splenocytes thatproduce antibodies against the HBsAg mutant from said rabbit with cellsfrom a rabbit plasmacytoma; (c) selecting for cells that secrete saidantibodies.
 61. A polynucleotide encoding a rabbit monoclonal antibodyor an immunoreactive fragment thereof, according to claim
 1. 62. Thepolynucleotide of claim 61, wherein the immunoreactive fragment encodedby the polynucleotide is a Fab, F(ab′)₂, Fv or an sFv fragment.